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CAP1-Mediated Actin Cycling Via ADF/Cofilin Proteins Is Essential for Asymmetric Division in Mouse Oocytes Zhe-Long Jin, Yu-Jin Jo, Suk Namgoong* and Nam-Hyung Kim*

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CAP1-Mediated Actin Cycling Via ADF/Cofilin Proteins Is Essential for Asymmetric Division in Mouse Oocytes Zhe-Long Jin, Yu-Jin Jo, Suk Namgoong* and Nam-Hyung Kim* © 2018. Published by The Company of Biologists Ltd | Journal of Cell Science (2018) 131, jcs222356. doi:10.1242/jcs.222356 RESEARCH ARTICLE CAP1-mediated actin cycling via ADF/cofilin proteins is essential for asymmetric division in mouse oocytes Zhe-Long Jin, Yu-Jin Jo, Suk Namgoong* and Nam-Hyung Kim* ABSTRACT 2013; Sun et al., 2011b) represent a family of proteins that initiate Dynamic reorganization of the actin cytoskeleton is fundamental to new actin filament formation and contribute to off-center meiotic a number of cellular events, and various actin-regulatory proteins spindle positioning. Whereas FMN2 and SPIRE are mainly involved modulate actin polymerization and depolymerization. Adenylyl in the formation of the cytoplasmic actin mesh network, the ARP2/3 cyclase-associated proteins (CAPs), highly conserved actin complex (Chaigne et al., 2015, 2013; Sun et al., 2011b), JMY monomer-binding proteins, have been known to promote actin (Sun et al., 2011a) and N-WASP (Yi et al., 2011) are responsible disassembly by enhancing the actin-severing activity of the for the cortical actin cap and subcortical actin network formation. ADF/cofilin protein family. In this study, we found that CAP1 In addition to actin nucleators, other actin-binding proteins, including regulated actin remodeling during mouse oocyte maturation. capping proteins (Jo et al., 2015), tropomodulin (Jo et al., 2016), Efficient actin disassembly during oocyte maturation is essential tropomyosins (Jang et al., 2014), and the actin depolymerizing for asymmetric division and cytokinesis. CAP1 knockdown impaired factor (ADF)/cofilin protein family, have emerged as important meiotic spindle migration and asymmetric division, and resulted in factors in cytoplasmic actin mesh network maintenance in mouse an accumulation of excessive actin filaments near the spindles. oocytes. In contrast, CAP1 overexpression reduced actin mesh levels. Modulation of the dynamic balance between filamentous actin CAP1 knockdown also rescued a decrease in cofilin family protein (F-actin) and globular actin (G-actin) is a central mechanism of actin overexpression-mediated actin levels, and simultaneous expression cytoskeleton regulation. To recycle actin monomers, the F-actin of human CAP1 (hCAP1) and cofilin synergistically decreased filament is depolymerized to form G-actin. Filament severing is cytoplasmic actin levels. Overexpression of hCAP1 decreased the mediated by ADF/cofilin family proteins, which bind to the sides of amount of phosphorylated cofilin, indicating that CAP1 facilitated filaments (Bernstein and Bamburg, 2010). In addition to ADF/cofilin actin depolymerization via interaction with ADF/cofilin during mouse proteins, several other proteins are involved in efficient actin filament oocyte maturation. Taken together, our results provide evidence for recycling. One type of protein involved in the actin-severing pathway the importance of dynamic actin recycling by CAP1 and cofilin in the is the adenylyl cyclase-associated protein (CAP, also known as SRV2 asymmetric division of mouse female gametes. in yeast) (Freeman and Field, 2000). CAP was first identified as a component of the yeast adenylyl cyclase complex that regulates This article has an associated First Person interview with the first both the actin cytoskeleton and the Ras/cAMP pathway (Fedor- author of the paper. Chaiken et al., 1990; Field et al., 1990). A recent study showed that CAPs enhanced actin monomer recharging with ATP antagonistically KEY WORDS: Actin, CAP1, ADF/cofilin, Oocyte, Asymmetric division to ADF/cofilin (Ono, 2013), and they also promoted the severing of actin filaments in cooperation with ADF/cofilin (Chaudhry et al., INTRODUCTION 2013). Yeast and mammalian CAP homologues enhanced actin The actin cytoskeleton consists of highly dynamic filament arrays, filament severing by 4–8-fold (Chaudhry et al., 2013; Jansen et al., whose assembly and rapid turnover are essential during various 2014) stages of mammalian oocyte maturation, including asymmetric Two distinct mammalian CAP isoforms are known, CAP1 spindle migration, cortical actin cap formation, polar body extrusion and CAP2 (Hubberstey and Mottillo, 2002; Yu et al., 1994, 1999). and cytokinesis (Almonacid et al., 2014; Sun and Schatten, 2006; In mammals, CAP1 is expressed ubiquitously, excluding in the Yi and Li, 2012). Various actin-binding proteins play essential roles skeletal muscle, and CAP2 is predominantly expressed in the brain, in the regulation of dynamic actin filament formation, elongation heart, skeletal muscle and testes (Yu et al., 1994). Human CAP1 has and depolymerization (Pollard and Borisy, 2003; Pollard and been found to play a key role in accelerating actin filament turnover Cooper, 2009). For example, actin nucleators such as formin-2 by effectively recycling cofilin family proteins (cofilin hereafter) (FMN2) (Dumont et al., 2007), SPIRE (Pfender et al., 2011) and and actin and by interacting with both ends of the actin filament the ARP2/3 complex (also known as ARPC) (Chaigne et al., 2015, (Moriyama and Yahara, 2002). CAP1 depletion leads to alterations in the actin cytoskeleton, cofilin and FAK phosphorylation, as well as increased cell adhesion and motility, and abnormal morphology Department of Animal Sciences, Chungbuk National University, Cheongju 361-763, in various cells (Bertling et al., 2004; Zhang et al., 2013). Mouse Korea. CAP1 forms hexameric structures that autonomously bind to F-actin, *Authors for correspondence ([email protected]; enhance cofilin-mediated severing, and catalyze the nucleotide [email protected]) exchange of actin (Jansen et al., 2014). Considering the importance of CAPs in the actin dynamics of Z.-L.J., 0000-0002-3408-0649; Y.-J.J., 0000-0002-9067-3537; S.N., 0000-0002- 4395-5558; N.-H.K., 0000-0003-1741-9118 several cells, we investigated the role of CAP1 in the actin dynamics of murine oocyte maturation, where dynamic actin organization is Received 5 July 2018; Accepted 23 October 2018 crucial for spindle migration and asymmetric divisions. Journal of Cell Science 1 RESEARCH ARTICLE Journal of Cell Science (2018) 131, jcs222356. doi:10.1242/jcs.222356 RESULTS meiotic maturation, we depleted CAP1 mRNA by introducing CAP1 localized in the cytoplasm and at the cortical actin cap CAP1 siRNA into oocytes. We confirmed via western blotting during mouse oocyte maturation (1.21±0.1 vs 0.52±0.4; mean±s.e.m.), immunostaining, and qPCR To investigate the functional role of CAP1 in mouse oocyte analyses (1.04±0.04 vs 0.12±0.05) that siRNA introduction maturation, we first examined CAP1 subcellular localization during caused CAP1 mRNA and protein depletion (Fig. 2A–C). CAP1 maturation. Oocytes were sampled at each developmental stage, knockdown (CAP1 KD) oocytes frequently exhibited multiple corresponding to the mature germinal vesicle stage (GV), germinal polar bodies, abnormally large polar bodies, or membrane bleb vesicle breakdown (GVBD), metaphase I (MI) and metaphase II (Fig. 2D,E). Co-injection of mScarlet–hCAP1 was shown to rescue (MII). Subcellular localization of endogenous CAP1 was monitored these defects (Fig. 2D–H), with abnormal polar body extrusion via immunofluorescence staining. CAP1 showed punctate distribution rates of: control, 6.06±3.03%; CAP1 KD, 31.72±3.51%; rescue: throughout the entire cytoplasm during all oocyte developmental 13.92±3.08% (Fig. 2G). Subsequently, we examined the effect of stages (Fig. 1A). CAP1 was also enriched at the cortical actin cap CAP1 knockdown on mouse oocyte maturation. The maturation during the MI stage (Fig. 1B; Fig. S1). To confirm CAP1 localization, rate of oocytes injected with CAP1 siRNA was lower than that of we expressed exogenous mScarlet-fused human CAP1 (mScarlet– control oocytes. The percentage of oocytes that matured to the MII hCAP1) in MI mouse oocytes. mScarlet–hCAP1 was dispersed stage was significantly decreased in the CAP1-knockdown oocytes throughout the cytoplasm and at the actin cap (Fig. 1C), supporting (Fig. 2I,J), with polar body extrusion rates of: control, 87.62±4.72%; the immunostaining localization results. CAP1 KD, 58.87±8.1%; rescue, 81.89±1.1% (Fig. 2J). Moreover, the ratio of polar body diameter to oocyte diameter was significantly Knockdown of CAP1 induced meiotic arrest and impaired higher in the CAP1 knockdown oocytes, which was also rescued asymmetric division of oocytes by mScarlet–hCAP1 injection (Fig. 2K). An abnormally large polar Previously, CAP1 depletion in somatic cells has been shown to lead body was defined as having a diameter 50% greater than that of the to F-actin accumulation and cytoskeletal defects (Zhang et al., oocyte. Collectively, these results indicated that CAP1 depletion 2013). Therefore, to investigate the function of CAP1 during mouse negatively impacted mouse oocyte asymmetric division. Fig. 1. CAP1 localization during mouse meiotic maturation. (A) Subcellular CAP1 localization during mouse oocyte meiosis, determined through staining with anti-CAP1 antibody. For the negative control, the anti-CAP1 primary antibody was omitted. CAP1 showed punctate distribution throughout the entire cytoplasm at all developmental stages. Blue, DNA; red, actin; green, CAP1. Scale bar: 20 μm. (B) Localization of CAP1 at the cortical actin cap in MI oocytes. Blue, DNA; red, actin; gray, CAP1. Scale bar: 10 μm. (C) Localization of mScarlet–human CAP1 (mScarlet–hCAP1) at MI oocytes. CAP1 was co-localized with actin in the cortical actin cap. Blue, DNA; green, actin; red, CAP1. Scale
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